At5g20260 Antibody
Description
Introduction to At5g20260 Antibody
The At5g20260 Antibody is a research-grade reagent targeting the protein encoded by the At5g20260 gene locus in Arabidopsis thaliana (mouse-ear cress). This antibody is primarily used in plant biology studies to detect and analyze the expression, localization, or function of the corresponding protein. The product is commercially available as a purified antibody solution (2ml/0.1ml) with a catalog number CSB-PA665587XA01DOA .
Mechanism of Action
-
Binding Specificity: Targets the At5g20260 protein with high affinity, enabling detection in assays.
-
Applications:
General Challenges in Antibody Research
-
Specificity: Cross-reactivity with homologous proteins in other plant species or subcellular compartments is a potential concern .
-
Avidity: Binding strength may vary depending on assay conditions (e.g., buffer pH, temperature) .
-
Immunogenicity: While not reported for this antibody, plant proteins can trigger immune responses in experimental models .
Comparative Analysis with Other Plant Antibodies
Below is a comparison of At5g20260 with other Arabidopsis-targeted antibodies from Cusabio’s catalog :
| Gene Locus | Uniprot No. | Potential Function | Applications |
|---|---|---|---|
| At5g20260 | Q3E9A4 | Unknown (requires validation) | Protein detection, localization |
| FRB2 | Q3EBQ3 | Possibly involved in stress response | Western blot, IHC |
| FRO5 | Q9FLW2 | Flavin-containing monooxygenase | Enzyme activity studies |
| FRO7 | Q3KTM0 | Oxidoreductase activity | Metabolic pathway analysis |
Future Directions and Recommendations
-
Validation Studies:
-
Functional Insights:
-
Investigate At5g20260’s role in metabolic pathways or stress responses using RNAi/CRISPR knockouts.
-
-
Cross-Species Utility:
Product Specs
Composition: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
FAQs for At5g20260 Antibody in Academic Research
Compiled based on current methodologies in antibody development and validation from peer-reviewed studies.
How can I validate the specificity of At5g20260 antibody in Arabidopsis thaliana experiments?
Methodological Answer:
-
Knockout Validation: Use CRISPR-edited Arabidopsis lines lacking At5g20260. Compare antibody binding in wild-type vs. knockout plants via Western blot or immunofluorescence .
-
Cross-Reactivity Testing: Include phylogenetically related species (e.g., Brassica napus) to assess off-target binding.
-
Epitope Mapping: Synthesize overlapping peptide fragments of At5g20260 and test antibody binding (see for peptide design workflows).
| Validation Step | Expected Outcome | Common Pitfalls |
|---|---|---|
| Western Blot | Single band at predicted molecular weight (~X kDa) | Non-specific bands due to glycosylation |
| Immunoprecipitation | Co-purification of known interactors (e.g., [hypothetical protein partners]) | Contamination from lysate debris |
| Tissue-Specific Staining | Localization matching transcriptomic data (e.g., root vs. leaf) | Autofluorescence in chloroplasts |
What experimental conditions optimize At5g20260 antibody performance in immunoassays?
Methodological Answer:
-
Buffer Optimization: Test pH (6.0–8.0) and ionic strength (50–200 mM NaCl) to minimize non-specific interactions .
-
Blocking Agents: Compare bovine serum albumin (BSA) vs. casein for signal-to-noise reduction.
-
Fixation Methods: For microscopy, evaluate paraformaldehyde (4%) vs. methanol fixation for antigen preservation.
How to resolve contradictions in At5g20260 subcellular localization studies?
Methodological Answer:
-
Multi-Method Corroboration: Combine:
-
Contextual Variables: Assess environmental stressors (e.g., drought, pathogens) that may alter localization dynamically.
Case Study from Literature:
In SARS-CoV-2 antibody studies, bispecific designs required iterative validation across >10 variants to confirm conserved epitopes . Apply similar rigor for plant stress responses.
What computational tools predict At5g20260 antibody-antigen binding dynamics?
Methodological Answer:
-
Structural Modeling: Use AlphaFold2 to predict At5g20260’s 3D structure and dock antibody paratopes .
-
Affinity Prediction: Tools like DyAb (see ) analyze mutational scans to forecast binding energy changes ().
-
Evolutionary Conservation Analysis: Identify invariant regions using PhyloP across 50+ plant genomes to prioritize stable epitopes.
| Tool | Application | Data Input Requirements |
|---|---|---|
| PyMOL | Visualization of docking results | PDB files of antibody-antigen complex |
| HADDOCK | Refinement of binding interfaces | NMR or cryo-EM constraints |
| RosettaAntibody | Design of humanized variants | Heavy/light chain sequences |
How to design a longitudinal study tracking At5g20260 expression across developmental stages?
Methodological Answer:
-
Sampling Protocol: Collect tissues at 7–10 day intervals from germination to senescence. Include root, leaf, and floral organs.
-
Normalization Controls: Use housekeeping genes (e.g., Actin2) and total protein quantification (Bradford assay) .
-
Data Integration: Pair antibody-based protein levels with RNA-seq data to identify post-transcriptional regulation.
Example Workflow from :
In COVID-19 antibody research, longitudinal mouse models tested efficacy against evolving variants. Adapt this framework with Arabidopsis phenotyping platforms.
Key Notes for Researchers
-
Antibody Revalidation: Repeat validation annually or after protocol changes (e.g., new imaging systems).
-
Negative Controls: Always include:
-
Pre-immune serum from the same host species
-
Secondary antibody-only samples.
-
-
Data Transparency: Publish raw blot images and microscopy metadata in repositories like Zenodo.
